Basic characteristics of calretinin (CR) and related calcium binding proteins identified under the project are used to learn about intracellular sites and modes of action. Recently published studies have revealed: (1) CR undergoes calcium-dependent conformational changes as shown by changes in intrinsic fluorescence and sites of digestion by trypsin (2) Analyses of four recombinant fragments of CR each containing 2-3 of the 6 potential calcium binding sites revealed calcium binding domains (EF hands) with high (Kd 0.4 muM) calcium affinities showing cooperative binding and calcium dependent conformational changes. (3) Subcellular fractionation studies showed CR and calbindin D28k are associated with synaptic and microsomal membrane fractions of rat cerebellum with greater association in the presence of calcium. The related calcium binding protein, parvalbumin, was only detected in the cytosolic fraction. (4) Polyclonal and monoclonal calcium binding protein antibodies preferentially recognize specific calcium-induced conformational states. Antibodies specific to CR and calbindin D28k recognized a Ca2+-bound form to a greater extent than the Ca2+-free form (either unfixed or following formalin treatment). Antisera produced in a rabbit from CR fixed in the calcium free form (with formalin) revealed immunoreactivity on brain sections that differed from that of antisera which preferentially recognize calcium bound CR. These data indicate that the calcium status of neurons during fixation can influence subsequent immunohistochemical staining by antibodies to calcium binding proteins and that conformation-specific antisera may be useful tools for studying the functional state of neurons.
